Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism, but is presently the prototypic osteoanabolic hormone for treating osteoporosis. Nevertheless, the anabolic window for PTH treatment limits its usefulness. As a result, other anabolic therapies are under investigation. These include PTHrP and its analog, abaloparatide. Both are thought to be superior to teriparatide (hPTH1-34) for treating osteoporosis. All three agents act through PTHR1 coupled to Gs? resulting in increases in cAMP. The histone deacetylase, HDAC4 (a repressor of transcription), has been shown to be a major mediator of PTHR1 activity in both bone and cartilage. Haploinsufficiency of HDAC4 in humans results in brachydactyly (short digits), as do mutations of PTHrP and the protein kinase A (PKA) regulatory subunit, PRKAR1A, indicating likely epistasis in this pathway. In osteoblasts, PTH causes exit of HDAC4 from the nuclei and derepression of transcription of catabolic genes, while PTHrP does not. Thus, the two hormones appear to have different actions on HDAC4. Our work in the last cycle of this grant led us to conclude that PTH action on the osteoblast causes PKA- dependent phosphorylation of HDAC4 in the nucleus resulting in its dissociation from Runx2 on the matrix metalloproteinase-13 (MMP-13) promoter and induction of MMP-13 transcription. PTH also causes PP2A- dependent nuclear dephosphorylation of HDAC4 at a different serine. The post-translationally modified HDAC4 then traffics from the nucleus to the cytoplasm and is ultimately degraded. Deletion of HDAC4 from mature osteoblasts in mice in vivo results in a mild bone phenotype with increased bone MMP-13, sclerostin and RANKL and enhanced PTH responsiveness. From these data of cells in culture and our preliminary data in vivo, we have developed the central hypothesis that phosphorylation of HDAC4 is an essential regulator of its function in the skeleton, and PTH or PTHrP regulates this process. The long-term goals of this work are to delineate the signaling and transcriptional regulatory mechanisms conveying PTH or PTHrP action in bone. Consequently, the specific aims to test our hypothesis of this resubmitted competing continuation proposal focus on the phosphorylation of HDAC4 and the role of PKA, and will, 1) Determine the mechanism of PTH, PTHrP or abaloparatide regulation of HDAC4 in osteoblasts by, a. examining how PKA and PP2A regulate nuclear HDAC4, b. defining how PP2A is regulated by PTH, PTHrP or abaloparatide, 2) Determine the effect of manipulation of PKA in mice on HDAC4, its regulated genes in bone and subsequent bone development and metabolism by, a. establishing the effect of increased activity of PKA in osteoblasts on skeletal phenotype, b. identifying the effect of decreased activity of PKA in osteoblasts on skeletal phenotype, c. comparing the effects of PTH, PTHrP or abaloparatide on mice with osteoblast-specific decrease in PKA activity. The results of this work will make the firs and major contributions to our knowledge of how the PTHR1 exerts its nuclear effects on skeletal function through PKA. Moreover, it will define how phosphorylation of HDAC4 contributes to this and provide information as to why PTHrP and abaloparatide show superiority to teriparatide. In so doing, the data will provide new perspectives into treatment of osteoporosis.